The chromatin remodelling factor and histone chaperone Yta7 is a member of the ATAD2 family of AAA+ ATPases from Saccharomyces cerevisiae that has in vivo functions consistent with both nucleosome assembly and disassembly activity. At the centromere, Yta7 is required for proper deposition of the centromeric histone H3 variant CENP-A Cse4. Here, we performed a genetic screen to identify suppressors of the defect of a mutation in CENP-A Cse4 that impairs the interaction with the DNA of the centromeric nucleosome (cse4-S135A). This identified two suppressor alleles of YTA7, yta7-R483S and -D518E, which are in the AAA1 domain of Yta7. Interestingly, Yta7-R483S enhanced the deposition of CENP-A Cse4 at the centromere and showed a ∼40% increased ATPase activity, suggesting that the hyperactivity of the motor domain is responsible for suppression of the cse4-S135A growth defect. In contrast, Yta7-D518E showed reduced ATPase activity, but both Yta7-R483S and -D518E retained the interaction with CENP-A Cse4 and centromeric sequences as well as hexamer formation in vitro. Our analysis of in vivo interactions between Yta7 and CENP-A Cse4 further showed that the two AAA+ domains and the non-canonical bromodomain of Yta7 are necessary and sufficient for interaction with CENP-A Cse4. The genetic screen furthermore revealed a mutation in the chromatin remodeler Fun30 as a suppressor of the centromeric defect of cse4-S135A. Altogether, this work reveals unusual, hypermorphic properties of Yta7 variants and highlights the importance of nucleosome remodelers in establishing centromeric chromatin.

Targeted Protein Deliveries In article number 2204620, Hak-Sung Kim and co-workers develop a new type of protein self-assembly using human clathrin for a targeted protein delivery. A redesigned clathrin triskelion is dual-functionalized with functional biomolecules. The resulting clathrin assembly exhibits a significantly high avidity and an efficient delivery of a protein cargo into tumor cells.

A stimuli-responsive protein self-assembly offers promising utility as a protein nanocage for biotechnological and medical applications. Herein, the development of a virus-like particle (VLP) that undergoes a transition between assembly and disassembly under a neutral and acidic pH, respectively, for a targeted delivery is reported. The structure of the bacteriophage P22 coat protein is used for the computational design of coat subunits that self-assemble into a pH-responsive VLP. Subunit designs are generated through iterative computational cycles of histidine substitutions and evaluation of the interaction energies among the subunits under an acidic and neutral pH. The top subunit designs are tested and one that is assembled into a VLP showing the highest pH-dependent structural transition is selected. The cryo-EM structure of the VLP is determined, and the structural basis of a pH-triggered disassembly is delineated. The utility of the designed VLP is exemplified through the targeted delivery of a cytotoxic protein cargo into tumor cells in a pH-dependent manner. These results provide strategies for the development of self-assembling protein architectures with new functionality for diverse applications.

The chromatin remodelling factor and histone chaperone Yta7 is a member of the ATAD2 family of AAA+ ATPases from Saccharomyces cerevisiae that has in vivo functions consistent with both nucleosome assembly and disassembly activity. At the centromere, Yta7 is required for proper deposition of the centromeric histone H3 variant CENP-A Cse4. Here, we performed a genetic screen to identify suppressors of the defect of a mutation in CENP-A Cse4 that impairs the interaction with the DNA of the centromeric nucleosome (cse4-S135A). This identified two suppressor alleles of YTA7, yta7-R483S and -D518E, which are in the AAA1 domain of Yta7. Interestingly, Yta7-R483S enhanced the deposition of CENP-A Cse4 at the centromere and showed a ∼40% increased ATPase activity, suggesting that the hyperactivity of the motor domain is responsible for suppression of the cse4-S135A growth defect. In contrast, Yta7-D518E showed reduced ATPase activity, but both Yta7-R483S and -D518E retained the interaction with CENP-A Cse4 and centromeric sequences as well as hexamer formation in vitro. Our analysis of in vivo interactions between Yta7 and CENP-A Cse4 further showed that the two AAA+ domains and the non-canonical bromodomain of Yta7 are necessary and sufficient for interaction with CENP-A Cse4. The genetic screen furthermore revealed a mutation in the chromatin remodeler Fun30 as a suppressor of the centromeric defect of cse4-S135A. Altogether, this work reveals unusual, hypermorphic properties of Yta7 variants and highlights the importance of nucleosome remodelers in establishing centromeric chromatin.

Targeted Protein Deliveries In article number 2204620, Hak-Sung Kim and co-workers develop a new type of protein self-assembly using human clathrin for a targeted protein delivery. A redesigned clathrin triskelion is dual-functionalized with functional biomolecules. The resulting clathrin assembly exhibits a significantly high avidity and an efficient delivery of a protein cargo into tumor cells.

A stimuli-responsive protein self-assembly offers promising utility as a protein nanocage for biotechnological and medical applications. Herein, the development of a virus-like particle (VLP) that undergoes a transition between assembly and disassembly under a neutral and acidic pH, respectively, for a targeted delivery is reported. The structure of the bacteriophage P22 coat protein is used for the computational design of coat subunits that self-assemble into a pH-responsive VLP. Subunit designs are generated through iterative computational cycles of histidine substitutions and evaluation of the interaction energies among the subunits under an acidic and neutral pH. The top subunit designs are tested and one that is assembled into a VLP showing the highest pH-dependent structural transition is selected. The cryo-EM structure of the VLP is determined, and the structural basis of a pH-triggered disassembly is delineated. The utility of the designed VLP is exemplified through the targeted delivery of a cytotoxic protein cargo into tumor cells in a pH-dependent manner. These results provide strategies for the development of self-assembling protein architectures with new functionality for diverse applications.

The assembly of proteins in a programmable manner provides insight into the creation of novel functional nanomaterials for practical applications. Despite many advances, however, a rational protein assembly with an easy scalability in terms of size and valency remains a challenge. Here, a simple bottom-up approach to the supramolecular protein assembly with a tunable size and valency in a programmable manner is presented. The dendrimer-like protein assembly, simply called a "protein dendrimer," is constructed through a stepwise and alternate addition of a building block protein. Starting from zeroth-generation protein dendrimer (pG₀ ) of 27 kDa, the protein dendrimer is sequentially grown to pG₁ , pG₂ , pG₃ , to pG₄ with a molecular mass of 94, 216, 483, and 959 kDa, respectively. The valency of the protein dendrimers at the periphery increases by a factor of two after each generation, allowing a tunable valency and easy functionalization. The protein dendrimers functionalizes with a targeting moiety and a cytotoxic protein cargo shows a typical feature of multi-valency in the avidity and a highly enhanced cellular cytotoxicity, exemplifying their utility as a protein delivery platform. The present approach can be effectively used in the creation of protein architectures with new functions for biotechnological and medical applications.

In eukaryotes, genomic DNA is packaged into hierarchical higher-order chromatin structures. Regulation of this organization is essential for the faithful maintenance and controlled expression of genetic information. Assembly of the nucleosome─the fundamental unit of chromatin─depends on a tightly coordinated network of histone chaperones that escort histones from their initial synthesis through multiple intermediates to their final deposition into nucleosomes. This review provides an overview of the stepwise journey of histones and the regulatory mechanisms that govern this process. We also highlight major outstanding questions and discuss future directions in the field.

Given the pivotal role of angiotensin-converting enzyme 2 (ACE2) in mediating viral entry and the genetic divergence observed in ACE2 orthologs across different species, we aimed to elucidate further the molecular intricacies underlying the interactions between severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike (S) protein and ACE2. In this study, we examined the amino acid residues in ACE2 orthologs interacting with SARS-CoV-2 spike receptor-binding domain to identify those with discernible effects on viral binding and entry. Through <i>in vitro</i> mutagenesis and <i>in silico</i> modeling studies of ACE2 variants, we have pinpointed the amino acid substitutions in human ACE2 that affect SARS-CoV-2 binding and entry. This work can significantly advance our understanding of the molecular mechanisms of SARS-CoV-2-host interactions, receptor recognition, viral entry process, and potential therapeutic options targeting coronavirus entry.